Phosphoenolpyruvate carboxykinase has been isolated from pig liver mitochondria and purified 208-fold from the initial mitochondrial accectone powder extract in good yield (48%). The enzyme appears homogeneous in sedimentation velocity experiments and has a sedimentation coefficient (s₂₀⁰,w) of 5.21 S. Its molecular weight, determined by sedimentation equilibrium is 73,300. The ultraviolet and visible absorption spectra of the enzyme revealed no characteristic chromophores. The amino acid composition of the enzyme is reported. Fifteen sulfhydryl groups per molecule of phosphoenolpyruvate carboxykinase account for its total half-cystine content. The N-terminal groups of phosphoenolpyruvate carboxykinase were found to be glutamic acid and isoleucine by Sanger's method. Substrate binding investigated by equilibrium dialysis indicates that PEP binding by phosphoenolpyruvate carboxykinase requires Mn⁺⁺, white GDP or GTP binding does not require the metal ion. K_s values determined by equilibrium dialysis were 2.3 x 10⁻⁶ M for PEP, and 3.6 x 10⁻⁶ M for GDP and 3.4 x 10⁻⁶ M for GTP. At 30°mitochondrial P-enolpyruvate carboxykinase catalyzes the inosine diphosphate- and Mn⁺⁺ - dependent carboxylation of 664 moles of phosphoenolpyruvate per min per mole of enzyme at pH 7.0 and the ITP- and Mn⁺⁺ - dependent decarboxylation of 4650 moles of oxalacetate per min per mole of enzyme at pH 7.5. The enzyme exhibits a relatively high degree of specificity for inosine and guanosine nucleotides in the carboxylation, decarboxylation and oxalacetate-H¹⁴CO₃⁻ exchange reactions. While purified IDP cannot replace ITP in the oxalacetate-H¹⁴CO₃⁻ exchange reaction, IDP can replace ITP in the decarboxylation reaction. In contrast to ITP-supported decarboxylation, IDP-supported decarboxylation leads to pyruvate rather than P-enolpyruvate formation. The enzyme is inhibited by low concentrations (10⁻⁶ M) of p-chloromercuribenzoate which can be reversed by high concentrations of glutathione. Comparative kinetic studies at pH 6.8, 7.3, and 8.0 reveal that the phosphoenolpyruvate carboxykinase-catalyzed inosine triphosphate-dependent oxalacetate-H¹⁴CO₃⁻ exchange is much more rapid than either the overall decarboxylation or carboxylation reactions. At pH 6.8, the relative carboxylation, decarboxylation, and oxalacetate-H¹⁴CO₃⁻ exchange rates are 1.0, 8.3, and 30, respectively. Under conditions which permit rapid P-enolpyruvate carboxykinase-catalyzed oxalacetate-H¹⁴CO₃⁻ exchange, neither GTP-GDP-8-¹⁴C-, nor oxalacetate-P-enolpyruvate-1-¹⁴C-exchange occurs at a significant rate. The inability of carboxykinase to catalyze these exchange reactions renders unlikely (a) the formation of phosphorylenzyme from GTP (or ITP) and enzyme as a step in the oxalacetate-H¹⁴CO₃⁻ exchange reaction or (b) the dissociation of either enzyme-P-enolpyruvate or enzyme-IDP (or GDP) as a rate-limiting step in the over-all decarboxylation reaction. A mechanism compatible with these results and other known characteristics of the reaction is presented. / Ph. D.